Two human osteoblast-like osteosarcoma cell lines show distinct expressionand differential regulation of parathyroid hormone-related protein

Parathyroid hormone (PTH)-related protein (PTHrP) acts as a local regulator of osteoblast function via mechanisms that involve PTH/PTHrP receptors lin ked to protein kinase A (PMA) and C (PKC). However, the regulation of PTHrP production and mRNA expression in human osteoblasts is poorly understood. Here we have characterized alternative PTHrP mRNA3' splicing variants, enco ding PTHrP isoforms of 139, 141, and 173 amino acids, and studied the regul ation of PTHrP and its mRNAs by activated PKA and PKC in two human osteobla st-like cell lines (KPDXM and TPXM). Using exon-specific Northern analysis and reverse transcriptase-coupled polymerase chain reaction, we identified mRNAs encoding PTHrP(1-139) and PTHrP(1-141) in both cell lines. PTHrP(1-13 9) mRNAs predominated in TPXM cells and PTHrP(1-173) mRNAs were only detect ed in TPXM[ cells. Activation of PKA or PKC resulted in different effects o n PTHrP and its mRNAs in the two cell lines. In TPXM cells, peptide-specifi c immunoassays detected high basal levels of PTHrP, increasing by 2-fold in cell extracts and 4-fold in culture media at 7h and 24 h after exposure to forskolin, respectively, paralleling changes in PTHrP mRNA expression. Pho rbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), a PKC activator, ha d no effect. In KPDXM cells, PTHrP was not detected in culture media under basal experimental conditions, and barely detectable amounts were present i n cell extracts of TPA-treated cells, although the mRNA levels increased su bstantially in response to TPA. In the responsive cell lines, the effects o n mRNA levels were dose dependent, and increased by 6.9- to 10.5-fold and 2 .0- to 4.1-fold at 4 h in TPXM and KPDXM cells after exposure to 10 mu M fo rskolin and 150 nM TPA, respectively. PTHrP mRNA levels then declined but w ere sustained above controls also at 12 h in both cell lines, albeit at con siderably higher levels in TPXM cells. The different responsiveness to agen ts activating PKA- and PKC-dependent pathways may depend on the cellular st ate of differentiation, or alternatively, cancer cell line-specific defects . Our data demonstrating distinct differences in mRNA species and the amoun ts of PTHrP produced by the two cell lines as compared with roughly equival ent overall mRNA levels may suggest that post-transcriptional mechanisms pl ay an important role in limiting the production of intracellular and secret ed PTHrPs in human osteoblastic cells.